Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in immunosuppressed patients, especially recipients of solid organ or bone marrow transplants. HCMV infection of neonates is associated with deafness, mental retardation, and mortality. Although five anti-viral drugs (GCV, val-GCV, CDV, PFA, Foscarnet) have been approved and licensed by the FDA for use in patients with HCMV infection, all of them have limitations that preclude their long-term use, including poor oral bioavailability dose-related toxicity, and resistance. Furthermore, cross-resistance among the viral DNA polymerase inhibitors (GCV, val- GCV, CDV, PFA) is observed, and therefore, compounds targeting a different step in the viral replication cycle are of vital importance. Our strategy to develop an improved treatment for HCMV is to target the initial phase of virus infection, including virus attachment, fusion and immediate-early viral gene expression with small molecules. HCMV entry is a multi-step process that is initiated with its binding to cell surface proteins followed by a fusion event. The viral capsid is released into the cytosol and traffics to the nucleus where it is uncoated depositing the genome in the nucleus. Viral gene expression proceeds in a temporal expression pattern with immediate-early (IE), early (E) and late (L) gene expression. Two well-characterized IE proteins, IE1 and IE2, are essential for viral replication and for controlling downstream transcription factors. In preliminary studies, we developed and piloted a high-content screen for inhibitors of early events of HCMV replication. This screen validated the use of a reporter gene (YFP) fused to IE2 as a readout to measure inhibition of early steps in HCMV infection. However, the limitations of the screen include the use of the laboratory AD169 HCMV strain, and the assay is subject to fluorescence quenching and interference by compounds. Thus, we plan to redesign the screen by constructing a variant of the HCMV clinical strain TB40/E that expresses a chimeric fusion of the IE2 protein product with luciferase. This recombinant virus will be used in a cell-based high throughput screen that will measure both cell viability and luciferase expression in the same assay well. This screening strategy has the potential to identify small molecule inhibitors that target both cellular and vira proteins essential for initial stages of viral infection. The goal of this proposal is to identify, validate, and characterize small molecule inhibitors of the early stages of HCMV infection. In Phase I, we will screen e200,000 discrete small molecules. We will apply several secondary cell-based assays to eliminate nonspecific inhibitors and cytotoxic compounds. The antiviral activity of priority hits will be confirmed. We will also investigate the mechanisms of action and resistance of early leads. This work will be collaboration between Microbiotix and the Tortorella lab at Mount Sinai. In Phase II, we will chemically optimize the best candidates and demonstrate activity in vivo in animal infection models. The ultimate goal of this project is to develop these optimized inhibitors into clinical candidates for treating patients at high-risk for HCMV disease.